EP1155139B2 - Method for microbially producing l-valine - Google Patents

Abstract

The present invention relates to a process for the microbial production of L-valine in which the dihydroxy acid-synthase (ilvD) activity and/or the ilvD gene expression is intensified in a microorganism. As an alternative or in combination with this, the acetohydroxy acid-synthase (ilvBN) activity and isomeroreductase (ilvC) activity and/or the ilvBNC gene expression are intensified in a microorganism. The process according to the invention preferably makes use of microorganisms in which the activity of at least one enzyme that is involved in a metabolic pathway that reduces the formation of L-valine is weakened or eliminated. Thus, for instance, the process according to the invention preferably makes use of microorganisms having a defect mutation in the threonine dehydratase (ilvA) gene and/or a defect mutation in one or more genes of the pantothenate synthesis.

Description

The present invention relates to a method for the microbial production of L-valine according to claim 1 to 11 and to be used in the method, transformed microorganisms according to claim 12 to 15.

The amino acid L-valine is a commercially important product used in animal nutrition, human nutrition and medicine. There is therefore a general interest in providing improved processes for the production of L-valine.

Valine can be prepared by chemical synthesis or biotechnologically by fermentation of suitable microorganisms in suitable nutrient solutions. The advantage of biotechnological production by microorganisms lies in the formation of the correct stereoisomeric form, namely the L-form of valine free of D-valine.

Different types of bacteria, such as Escherichia coli, Serratia marcescens, Corynebacterium glutamicum, Brevibacterium flavum or Brevibacterium lactofermentum can produce L-valine in a nutrient solution containing glucose. US 5,658,766 shows that in Escherichia coli by mutation in the aminoacyl-tRNA synthetase increased formation can be achieved by L-valine. WO 96,06926 further shows that lipoic acid auxotrophy can increase L-valine formation with Escherichia coli. EP 0 694 614 A1 describes strains of Escherichia coli carrying resistance to α-ketobutyric acid and producing L-valine, L-isoleucine or L-leucine in a nutrient solution containing glucose.

In EP 0 477 000 it is shown that by mutagenesis of Corynebacterium or Brevibacterium and selection for valine resistance, L-valine formation can be improved. The same EP document also shows that by selecting Corynebacterium or Brevibacterium for resistance to various pyruvate analogs, such as β-fluoropyruvate, β-chloropyruvate, β-mercaptopyruvate or trimethylpyruvate, improved L-valine formation can be achieved. By Nakayama et al. (Nakayama et al., 1961, J. Gen. Appl. Microbiol. Jpn ), it is known that auxotrophies introduced by undirected mutations can lead to improved L-valine accumulation.

In EP 0 356 739 A1 In addition, it is shown that in amplification of the acetohydroxy acid synthase (ilvBN, see also FIG. 1 ) encoding DNA region by means of the plasmid pAJ220V3 the formation of L-valine is improved.

EP-A-872 547 discloses that in Escherichia microorganisms, the production of L-valine can be increased by a modification of the H ⁺ ATP gene. The gene may also include the IlvA gene.

Journal of Bacteriology, 1993, Vol. 175, No. 17, pp. 5595-5603 discloses that acetohydroxy acid synthase and isomeroreductase (ilvC) are catalysts for sequential reactions in the path to, inter alia, valine in Co rynebacterium glutamicum . Acetohydroxy acid synthase is encoded by two genes, ilvB and ilvN.

Nucleic Acids Research, 1987, Vol. 15, No. 5, pp. 2137-2155 discloses the sequencing of the ilvGMEDA operon of E. coli containing 5 genes which encode for 4 of the 5 enzymes necessary for the biosynthesis of L-valine.

Gene, 1993, Volume 137, pages 179-185 discloses sequencing of the ilv3 gene in Saccharomyces cerevisiae , which is necessary for dihydroxy acid hydratase biosynthesis.

The Japanese font Tokkai Hei 8-89249 discloses a coryneform bacterial DNA encoding dihydroxy acid dehydratase which can be used for the production of L-valine.

Japanese Tokkai Hei 5-344 893 shows that the production of L-valine can be enhanced by the use of plasmids carrying the gene of acetohydroxy acid synthase.

The publication "Isoleucine Synthesis in Corynebacterium glutamicum : Molecular Analysis of ilvB - ilvN - ilvC Operon" by Keilhauer et al., Journal of Bacteriology, Sept. 1993, p. 5595-5603 reveals structural analyzes of the ilvBNC operon.

The EP 1006 189 A2 discloses a process for the production of D-pantothenic acid in which the genes panB and panC are amplified individually or in combination with each other, in particular overexpressed. Optionally, a defect mutation of ilvA can be performed or an amplification or overexpression of the genes ilvBN, ilvD or ilvC can be performed.

It is an object of the present invention to provide new bases for the microbial production of L-valine, in particular with the aid of coryneform bacteria.

This object is achieved according to the invention in that the dihydroxy acid dehydratase (ilvD) activity and / or ilvD gene expression in a microorganism is enhanced, the activity of one or more enzymes specifically involved in the synthesis of D-pantothenate being attenuated or eliminated , In combination, acetohydroxy acid synthase (ilvBN) and isomeroreductase (ilvC) activity and / or ilvBNC gene expression in a microorganism is enhanced. In addition, microorganisms in which the activity of at least one enzyme involved in a metabolic pathway which decreases L-valine formation is attenuated or eliminated may be used for the methods of the present invention. For example, microorganisms having a defect mutation in the threonine dehydratase (ilvA) gene and / or having a defect mutation in one or more genes of the pantothenate synthesis are preferably used in the process according to the invention.

With the terms "valine" or "L-valine" in the sense of the claimed invention, not only the free acid, but also the salt thereof, such as. The calcium, sodium, ammonium or potassium salt.

The term "enhancement" describes the enhancement of the intracellular activity of said enzymes ilvD, ilvB, ilvN and ilvC. To increase the enzyme activity, in particular the endogenous activity in the microorganism is increased. An increase in the enzyme activity can be achieved, for example, in which, by changing the catalytic center, an increased substrate conversion takes place or in which the action of enzyme inhibitors will be annulled. Also, increased enzyme activity can be achieved by increasing enzyme synthesis, for example by gene amplification or by elimination of factors that repress enzyme synthesis. The endogenous enzyme activity is preferably increased according to the invention by mutation of the corresponding endogenous gene. Such mutations can be generated either undirected by conventional methods, such as UV irradiation or mutagenic chemicals, or targeted by genetic engineering methods, such as deletion (s), insertion (s) and / or nucleotide exchange (s).

The enhancement of gene expression according to the invention is preferably carried out by increasing the gene copy number. For this purpose, the gene or genes are incorporated into a gene construct or into a vector which preferably contains gene sequences associated regulatory gene sequences, in particular those that enhance gene expression. Subsequently, a microorganism, preferably Corynebacterium glutamicum, is transformed with the corresponding gene constructs.

It has been found that enhanced expression of the valine biosynthetic gene ilvD from Corynebacterium glutamicum, which codes for the enzyme dihydroxy acid dehydratase, produces L-valine in an improved manner. According to the invention, in addition to the increased expression of this gene, the enhanced expression of the ilvBN genes, which code for the enzyme acetohydroxy acid synthase, and of the ilvC gene, which codes for the enzyme isomeroreductase, in Corynebacterium glutamicum bring about improved L-valine formation. Further enhancement of L-valine formation is achieved by overexpression of all the genes mentioned in Corynebacterium glutamicum. The genes or gene constructs may be present in the host organism either in different copy number plasmids or integrated and amplified in the chromosome.

Further increase in gene expression may be effected, alternatively or in combination with an increase in gene copy number, by enhancing regulatory factors that positively affect gene expression. Thus, enhancement of regulatory elements at the transcriptional level can be achieved, in particular by using amplified transcription signals. Also, the promoter and regulatory region located upstream of the structural gene can be mutated. In the same way, expression cassettes act, which are installed upstream of the structural gene. By inducible promoters it is additionally possible to increase expression in the course of fermentative L-valine formation. In addition, however, an enhancement of the translation is possible, for example by the stability of the m-RNA is improved. Furthermore, genes can be used which code for the corresponding enzyme with a high activity. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture. Instructions for this, the expert finds among others Martin et al. (Bio / Technology 5, 137-146 (1987) ), at Guerrero et al. (Gene 138, 35-41 (1994) ) Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988) ), at Eikmanns et al. (Gene 102, 93-98 (1991) ), in the European Patent EP 0 472 869 , in the U.S. Patent 4,601,893 , at Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991) , at Reinscheid et al. (Applied and Environmental Microbiology 60,126-132 (1994) ), at LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993) ) and in the patent application WO 96/15246 ,

For amplification of gene expression, all possible combinations of the above measures are also possible.

Microorganisms which can be used in the process according to the invention can produce L-valine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It may be Gram-positive bacteria z. B. the genus Bacillus or coryneform bacteria of the already mentioned genus Corynebacterium or Arthrobacter act. In the genus Corynebacterium, in particular, the species Corynebacterium glutamicum has already been mentioned, which is known in the art for its ability to form amino acids. To this species are wild-type strains, such. B. Corynebacterium glutamicum ATCC13032, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869, Brevibacterium thiogenitalis ATCC19240, Corynebacterium molassecola ATCC17965 and others.

To isolate the gene ilvD of Corynebacterium glutamicum or other genes, a gene bank is first created. The creation of gene banks is written down in well-known textbooks and manuals. As an example, the textbook of Winnacker: Genes and Clones, An Introduction to Genetic Engineering (Verlag Chemie, Weinheim, Germany, 1990 ) or the manual of Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989 ) called. A known gene bank is that of the E. coli K-12 strain W3110, which is from Kohara et al. (Cell 50, 495-508 (1987) ) which was created in λ vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996 ) describe a gene bank of Corynebacterium glutamicum ATCC13032, which with the aid of the cosmid vector SuperCos I ( Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164 ) in E. coli K-12 NM554 ( Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575 ) was created. For the production of a gene bank of Corynebacterium glutamicum in Escherichia coli, plasmids such as pBR322 ( Bolivar, Life Sciences, 25, 807-818 (1979) ) or pUC19 ( Norrander et al., 1983, Gene, 26: 101-106 ) be used. For the preparation of a gene bank of Corynebacterium glutamicum in Corynebacterium glutamicum, plasmids such as pJC1 ( Cremer et al., Mol. Gen. Genet. (1990) 220: 3221-3229 ) or pECM2 ( Jäger et al., J. Bacteriol. (1992) 174: 5462-5465 ) be used. Hosts which are particularly suitable are those bacterial strains which are deficient in restriction and recombination. An example of this is the strain Escherichia coli DH5α mcr , which is from Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649 ), or the strain Corynebacterium glutamicum R127, of Liebl et al. was isolated (FEMS Lett (1989) 65: 299-304 ).

The gene bank is then transformed into an indicator strain by transformation ( Hanahan, Journal of Molecular Biology 166, 557-580, 1983 ) or electroporation ( Tauch et al., 1994, FEMS Microbiological Letters, 123: 343-347 ) built-in. The indicator strain is characterized by having a mutation in the gene of interest that elicits a detectable phenotype, eg, an auxotrophy. The indicator strains or mutants are available from published sources or Stammsamrnlungen or may need to be self-produced. In the context of the present invention, the Corynebacterium glutamicum mutant R127 / 7 has been isolated, which is defective in the ilvD gene coding for the dihydroxy acid dehydratase. After transformation of the indicator strain, such as ilvD mutant R127 / 7, with a recombinant plasmid carrying the gene of interest, such as the ilvD gene, and expression thereof, the indicator strain becomes the corresponding characteristic, such as L-valine Neediness, prototrophic.

The thus isolated gene or DNA fragment can be determined by determining the sequence, such as Sanger et al. (Proceedings of the United States of America of the United States of America, 74: 5463-5467, 1977 ) are characterized. Subsequently, the degree of identity to known genes found in databases such as GenBank ( Benson et al., 1998, Nuleic Acids Research, 26: 1-7 ) are analyzed with published methods ( Altschul et al., 1990, Journal of Molecular Biology 215: 403-410 ).

In this way, the DNA sequence coding for the gene ilvD was obtained from Corynebacterium glutamicum, which is part of the present invention as SEQ ID NO 1. Furthermore, the amino acid sequences of the corresponding enzymes were derived from the present DNA sequence with the methods described above. SEQ ID NO 2 shows the resulting amino acid sequence of the ilvD gene product, namely dihydroxy acid dehydratase.

The thus characterized gene can then be expressed individually or in combination with others in a suitable microorganism for expression. One known method of expressing or overexpressing genes is to amplify them by means of plasmid vectors, which may also be equipped with expression signals. Suitable plasmid vectors are those which can replicate in the corresponding microorganisms. For Corynebacterium glutamicum, for example, the vectors pEKEx1 ( Eikmanns et al., Gene 102: 93-98 (1991) ) or pZ8-1 ( European Patent 0 375 889 ) or pEKEx2 ( Eikmanns et al. Microbiology 140: 1817-1828 (1994) or pECM2 ( Jäger et al. Journal of Bacteriology 174 (16): 5462-5465 (1992) ) in question. Examples of such plasmids are pJC1ilvD, pECM3ilvBNCD, and pJC1ilvBNCD. These plasmids are Escherichia coli / Corynebacterium glutamicum shuttle vector carrying the gene ilvD or the gene ilvD together with the genes ilvB, ilvN, and ilvC.

The inventors have further found that the amplification of the gene, individually or in combination with the genes ilvB, ilvN and ilvC, has an advantageous effect in microorganisms which have a reduced synthesis of the amino acid L-isoleucine. This reduced synthesis can be achieved by deletion of the ilvA gene, which codes for the specific for L-isoleucine synthesis enzyme threonine dehydratase.

The deletion may be by directed recombinant DNA techniques. With the help of these methods, for example, the ilvA gene coding for the threonine dehydratase can be deleted in the chromosome. Suitable methods are included Schäfer et al. (Gene (1994) 145: 69-73 ) or too Link et al. (Journal of Bacteriology (1998) 179: 6228-6237 ). Also, only parts of the gene can be deleted, or mutated fragments of the Threonindehydratasegens be replaced. By deletion, a loss of Threonindehydrataseaktivität is achieved. An example of such a mutant is the Corynebacterium glutamicum strain ATCC13032ΔilvA, which carries a deletion in the ilvA gene.

The inventors have furthermore found that the amplification of the genes ilvD, ilvB, ilvN and ilvC in a further combination with the reduced synthesis of D-pantothenate, preferably in combination with further deletion of the ilvA gene, in microorganisms advantageously has an effect on the L- Valine formation affects, for example, by deletions in the panB and panC gene. The reduced D-pantothenate synthesis can be achieved by attenuation or elimination of the corresponding biosynthetic enzymes or their activities. For example, the enzymes ketopantoate hydroxymethyltransferase (EC 2.1.2.11), ketopantoate reductase, pantothenate ligase (EC 6.3.2.1) and aspartate decarboxylase (EC 4.1.1.11) are suitable for this purpose. One way to eliminate or mitigate enzymes and their activities is through mutagenesis.

These include undirected methods using chemical reagents, such as N-methyl-N-nitro-N-nitrosoguanidine, or UV irradiation for mutagenesis, followed by a search of the desired microorganisms for D-pantothenate need. Methods for mutation induction and mutant searches are well known and can be used, inter alia, in Miller (A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992 ) or in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington DC, USA, 1981).

Furthermore, this includes directed recombinant DNA techniques. With the aid of these methods, it is possible, for example, to delete the genes panB, panC, panE and panD coding for ketopantoate hydroxymethyltransferase, pantothenate ligase, ketopantoic acid reductase or aspartate decarboxylase individually or else together in the chromosome. Suitable methods are included Schäfer et al. (Gene (1994) 145: 69-73 ) or too Link et al. (Journal of Bacteriology (1998) 179: 6228-6237 ). Also, only parts of the genes can be deleted or mutated fragments of ketopantoate hydroxymethyltransferase, pantothenate ligase, ketopantoic acid reductase and aspartate decarboxylase can be exchanged. By deletion or replacement, a loss or a reduction of the respective enzyme activity is achieved. An example of such a mutant is the Corynebacterium glutamicum strain ATCC13032ΔpanBC, which carries a deletion in the panBC operon.

The microorganisms produced according to the invention can be cultured continuously or batchwise in the batch process (batch culturing) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of L-valine production. A summary of known cultivation methods are in the textbook of Chmiel (Bioprocessing Technology 1. Introduction to Bioprocess Engineering (Gustav Fischer Verlag, Stuttgart, 1991 )) or in the textbook of Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994 )).

The culture medium to be used must suitably satisfy the requirements of the respective microorganisms. Descriptions of culture media of various microorganisms are included in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C., USA, 1981). As the carbon source, sugars and carbohydrates such as e.g. Glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as. As soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such. As palmitic acid, stearic acid and linoleic acid, alcohols such. As glycerol and ethanol and organic acids such. As acetic acid can be used. These substances can be used individually or as a mixture. As the nitrogen source, organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may be used. The nitrogen sources can be used singly or as a mixture. As the phosphorus source, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used. The culture medium must further contain salts of metals, e.g. Magnesium sulfate or iron sulfate necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be used in addition to the above-mentioned substances. The said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation.

For pH control of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acidic compounds such as phosphoric acid or sulfuric acid are suitably used. To control foaming, antifoams, such as e.g. Fatty acid polyglycol esters. In order to maintain the stability of plasmids, suitable selective substances, e.g. Antibiotics, to be added. In order to maintain aerobic conditions, oxygen or oxygen containing gas mixtures, e.g. Air, registered in the culture. The temperature of the culture is usually 20 ° C to 50 ° C, and preferably 25 ° C to 45 ° C. The culture is continued until a maximum of L-valine has formed. This goal is usually reached within 10 hours to 160 hours.

The concentration of L-valine formed can be determined by known methods ( Jones and Gilligan (1983) Journal of Chromatography 266: 471-482 ).

The invention will be explained in more detail with reference to the following exemplary embodiments:

Example 1 Cloning, Sequencing and Expression of the Dihydroxy acid dehydratase-encoding ilvD gene from Corynebacterium glutamicum 1. Isolation of an ilvD mutant of Corynebacterium glutamicum

The strain Corynebacterium glutamicum R127 ( Haynes 1989, FEMS Microbiology Letters 61: 329-334 ) was mutagenized with N-methyl-N-nitro-N-nitrosoguanidine (Sambrook et al., Molecular Cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press). For this purpose, 5 ml of an overnight culture of Corynebacterium glutamicum were admixed with 250 μl of N-methyl-N-nitro-N-nitrosoguanidine (5 mg / ml dimethylformamide) and incubated for 30 minutes at 30 ° C. and 200 rpm ( Adelberg 1958, Journal of Bacteriology 76: 326 ). The cells were then washed twice with sterile NaCl solution (0.9%). By replica plating on minimal medium plates CGXII with 15 g / l agar ( Keilhauer et al., Journal of Bacteriology 175: 5595-5603 ) mutants were isolated, which grew only with the addition of L-valine, L-isoleucine and L-leucine (0.1 g / l each).

The enzyme activity of dihydroxy acid dehydratase was determined in the crude extract of these mutants. For this, the clones were cultured in 60 ml of LB medium and centrifuged off in the exponential growth phase. The cell pellet was washed once with 0.05 M potassium phosphate buffer and resuspended in the same buffer. The cell disruption was carried out by means of ultrasound treatment for 10 minutes (Branson Sonifier W-250, Branson Sonic Power Co, Danbury, USA). The cell debris was then separated by centrifugation at 13,000 rpm and 4 ° C. for 30 minutes, and the supernatant was used as the crude extract in the enzyme assay. The reaction batch of the enzyme test contained 0.2 ml of 0.25 M Tris / HCl, pH 8, 0.05 ml crude extract, and 0.15 ml of 65 mM alpha, beta-dihydroxy-beta-methylvalerate. The test mixtures were incubated at 30 ° C., after 10, 20 and 30 minutes 200 μl samples were taken and their ketomethylvalerate concentration was determined by HPLC analysis ( Hara et al. 1985, Analytica Chimica Acta 172: 167-173 ). As Table 1 shows, strain R127 / 7 has no dihydroxy-acid dehydratase activity, whereas the isomeroreductase and acetohydroxy acid synthase activities are still present as further branched-chain amino acid branching enzymes. Table 1 Specific activities (μmol / min and mg protein) of valine biosynthetic enzymes in Corynebacterium glutamicum strains tribe Dihydroxyacid dehydratase Isomero reductase Acetohydroxy acid synthase R127 0,003 0.05 0.07 R127 / 7 0,000 0.06 0.09

2. Cloning of the ilvD gene of Corynebacterium glutamicum

Chromosomal DNA from Corynebacterium glutamicum R127 was as in Schwarzer and Pühler (Bio / Technology 9 (1990) 84-87 ), isolated.

This was cleaved with the restriction enzyme Sau3A (Boehringer Mannheim) and purified by sucrose density gradient centrifugation ( Sambrook et al., Molecular cloning. A laboratory manual (1989) Cold Spring Harbor Laboratory Press ) separated. The fraction with the fragment size range of about 6-10 kb was used for ligation with the vector pJC1 (FIG. Cremer et al., Molecular and General Genetics 220 (1990) 478-480 ) used. The vector pJC1 was linearized for this purpose with BamHI and dephosphorylated. Five ng of these were ligated with 20 ng of said fraction of the chromosomal DNA and thus the mutant R127 / 7 by electroporation ( Haynes and Britz, FEMS Microbiology Letters 61 (1989) 329-334 ). The transformants were tested for the ability to grow on CGXII agar plates without addition of the branched-chain amino acids. Of more than 5,000 transformants tested, eight clones grew on minimal medium plates after replica plating and two days incubation at 30 ° C. Of these clones, plasmid preparations were made as in Schwarzer et al. (Bio / Technology (1990) 9: 84-87 ). Restriction analyzes of the plasmid DNA revealed that the same plasmid, referred to below as pRV, was present in all 8 clones. The plasmid carries a 4.3 kb insert and was retransformed for its ability to complement the ilvD mutant R127 / 7. By subcloning, the region responsible for the complementation of the mutant R127 / 7 was limited to a 2.9 ScaI / XhoI fragment ( FIG. 2 ).

3. Sequencing of the ilvD gene

The nucleic acid sequence of the 2.9 kb ScaI / XhoI fragment was prepared by the dideoxy chain termination method of Sanger et al. (Proceedings of the National of Sciences of the United States of America USA (1977) 74: 5463-5467 ). The Auto-Read sequencing kit was used (Amersham Pharmacia Biotech, Uppsala, Sweden). The gel electrophoretic analysis was carried out with the automatic laser fluorescence sequencer (ALF) from Amersham Pharmacia Biotech (Uppsala, Sweden). The obtained nucleotide sequence was analyzed with the program package HUSAR (Release 4.0, EMBL, Cambridge, UK). The nucleotide sequence is shown as ID SEQ NO 1. The analysis revealed an open reading frame of 1836 base pairs, identified as the ilvD gene, encoding a polypeptide of 612 amino acids, represented as SEQ ID NO 2.

4. Expression of the ilvD gene

The plasmid pRV was digested with the restriction enzymes ScaI and XhoI, according to the instructions of the manufacturer of the restriction enzymes (Roche, Boehringer Mannheim). Subsequently, the 2.9 kb ilvD fragment was isolated by Ionenaustauschersäulchen (Quiagen, Hilden). The overhanging end of the XhoI cut of the isolated fragment was filled in with Klenow polymerase. The vector pJC1 ( Cremer et al., Mol. Gen. Genet (1990) 220: 478-480 ) was Pst I cut, also treated with Klenow polymerase, and then ligated fragment and vector. With the ligation mixture, the E. coli strain DH5αmcr ( Grant et al., Proceedings of the National of Sciences of the United States of America, USA, 87 (1990) 4645-4649 ) ( Hanahan, Journal of Molecular Biology 166 (1983) 557-580 ). By plasmid preparations ( Sambrook et al., Molecular cloning. A laboratory manual (1989) Cold Spring Harbor Laboratory Press ) of clones, a clone containing the recombinant plasmid pJC1ilvD was identified. With this plasmid, Corvnebacterium glutamicum ATCC13032 was electroporated transformed, as in Haynes et al. (1989, FEMS Microbiol. Lett. 61: 329-334 ). Corynebacterium glutamicum ATCC13032 pJC1 and Corynebacterium glutamicum ATCC13032 pJClilvD were then used to determine ilvD-encoded dihydroxy-acid dehydratase activity. For this, the clones were cultured in 60 ml of LB medium and centrifuged off in the exponential growth phase. The cell pellet was washed once with 0.05 M potassium phosphate buffer and resuspended in the same buffer. The cell disruption was carried out by means of ultrasound treatment for 10 minutes (Branson Sonifier W-250, Branson Sonic Power Co, Danbury, USA). The cell debris was then separated by centrifugation at 13,000 rpm and 4 ° C. for 30 minutes, and the supernatant was used as the crude extract in the enzyme assay. The reaction assay of the enzyme assay contained 0.2 ml of 0.25 M Tris / HCl, pH 8, 0.05 ml crude extract, and 0.15 ml of 65 mM alpha, beta-dihydroxy-beta-methylvalerate. The test mixtures were incubated at 30 ° C., after 10, 20 and 30 minutes 200 μl samples were taken and their ketomethylvalerate concentration was determined by HPLC analysis ( Hara et al. 1985, Analytica Chimica Acta 172: 167-173 ). As shown in Table 2, strain Corynebacterium glutamicum ATCC13032 pJC1ilvD has increased dihydroxy acid dehydratase activity over the control strain. Table 2 Specific activity (μmol / min and mg protein) of dihydroxy acid dehydratase in Corynebacterium glutamicum ATCC13032 plasmid Dihydroxyacid dehydratase pJC1 0,008 pJC1ilvD 0,050

Example 2: Construction of an ilvA deletion mutant of Corynebacterium glutamicum

The internal deletion of the ilvA gene of Corynebacterium glutamicum ATCC13032 was with the Schäfer et al. (Gene 145: 69-73 (1994) ) performed gene exchange system. For the construction of the inactivation vector pK19mobsacBΔilvA, first of all the expression on an EcoRI fragment in the vector pBM21 (FIG. Möckel et al. 1994, Molecular Microbiology 13: 833-842 ) present ilvA gene removes an internal 241 bp BglII fragment. For this purpose, the vector was cut with BglII and, after separation of the ilvA internal BglII fragment by means of agarose gel electrophoresis, religated. Subsequently, the incomplete gene was isolated from the vector as an EcoRI fragment and inserted into the EcoRI linearized vector pK19mobsacB (FIG. Schäfer 1994, Gene 145: 69-73 ) ligated. The resulting inactivation vector pK19mobsacBΔilvA was introduced by transformation into the E. coli strain S 17-1 ( Hanahan 1983, Journal of Molecular Biology 166: 557-580 ) and conjugated to Corynebacterium glutamicum ATCC13032 ( Schäfer et al. 1990, Journal of Bacteriology 172: 1663-1666 ). Kanamycin-resistant clones of Corynebacterium glutamicum were obtained in which the inactivation vector was integrated in the genome. To select for excision of the vector, kanamycin resistant clones were seeded on sucrose-containing LB medium ( Sambrook et al., Molecular cloning. A laboratory manual (1989) Cold Spring Harbor Laboratory Press ) with 15 g / l agar, 2% glucose / 10% sucrose and obtained colonies which have lost the vector by a second recombination event ( Jäger et al. 1992, Journal of Bacteriology 174: 5462-5465 ). By inoculating on minimal medium plates (medium CGXII with 15 g / l agar ( Keilhauer et al., Journal of Bacteriology 175 (1993) 5595-5603 ) with and without 2 mM L-isoleucine or with and without 50 μg / ml kanamycin, 36 clones were isolated which were auxotrophic due to the excision of the vector kanamycin sensitive and isoleucine and in which the incomplete ilvA gene (ΔilvA allele) in the Genome was present. One strain was designated ATCC13032ΔilvA and was used further.

Example 3 Cloning of the Pantothenate Biosynthesis Genes panB and panC from C. glutamicum Cloning of the operon

Chromosomal DNA from C. glutamicum ATCC13032 was isolated and cut with the restriction endonuclease Sau3A. After gel electrophoretic separation, DNA fragments in the size range from 3 to 7 or from 9 to 20 kb were extracted and subsequently ligated into the singular BamHI site of the vector pBR322. Insert-bearing colonies were isolated by tetracycline sensitivity after inoculation on LB plates with 10 μg / ml tetracycline. Plasmid preparations (Sambrook et al., Molecular cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press) of pooled clones gave 8 plasmid pools, each containing 400 plasmids with an insert size of 9 to 20 kb and 9 plasmid pools, each containing 500 plasmids with an insert size of 3 to 7 kb, isolated. The E. coli panB mutant SJ2 ( Cronan et al. 1982, J. Bacteriol. 149: 916-922 ) was isolated with this gene bank by electroporation ( Wehrmann et al. 1994, Microbiology 140: 3349-3356 ). The transformation approaches were directly transferred to CGXII medium ( J. Bacteriol. (1993) 175: 5595-5603 ) plated. From clones capable of growth without pantothenate supplementation, plasmid DNA was isolated (Sambrook et al., 1989), and by retransformation, 8 clones were obtained whose D-pantothenate requirement was confirmed. Restriction mapping was performed on the 8 plasmids. One of the vectors studied, hereinafter called pUR1, contained an insert of 9.3 kb ( FIG. 3 ). The transformation of E. coli panC_Mutante DV39 ( Vallari et al. 1985, J. Bacteriol. 164: 136-142 ) revealed that the vector pUR1 was also able to complement the panC defect of this mutant. A 2.2 kb fragment of the insert of pUR1 was prepared by the dideoxy chain termination method of Sanger et al. sequenced ( Proc. Natl. Acad. Sci. USA (1977) 74: 5463-5467 ). The gel electrophoretic analysis was carried out with the automatic laser fluorescence sequencer (ALF) from Amersham Pharmacia Biotech (Uppsala, Sweden). The obtained nucleotide sequence was analyzed with the program package HUSAR (Release 4.0, EMBL, Cambridge, UK). The nucleotide sequence is shown as SEQ ID NO. 3 reproduced. The analysis revealed the identification of two open reading frames. An open reading frame comprises 813 base pairs and has high homologies to previously known panB genes from other organisms. The C. glutamicum panB gene encodes a polypeptide of 271 amino acids (see SEQ ID No. 4). The second open reading frame comprises 837 base pairs and has high homologies to already known panC genes from other organisms. The C. glutamicum panC gene encodes a polypeptide of 279 amino acids (see SEQ ID No. 5).

Example 4: Construction of a panBC deletion mutant of Corynebacterium glutamicum

The genomic panBC fragment of Corynebacterium glutamicum ATCC13032 and Corynebacterium glutamicum ATCC13032ΔilvA was used with the Schäfer et al. (Gene 145: 69-73 (1994) ) performed gene exchange system. To construct the deletion vector pK19mobsacBΔpanBC, the 3.95 kb SspI / SalI fragment was first ligated with panBC with pUC18, which had previously been cut into SmaI / SalI. Subsequently, a 1293 bp EcoRV / NruI fragment was removed from the overlapping region of the panBC genes by restriction digestion and religation. To allow for cloning in pK19mobsacB, the 2 primers 5'-GAGAACTTAATCGAGCAACACCCCTG, 5'-GCGCCACGCCTAGCCTTGGCCCTCAA and the polymerase chain reaction (PCR) were used to amplify the deleted panBC region in pUC18 to obtain a 0.5 kb ΔpanBC fragment at the ends a SaII, BEZW. EcoRI interface carries. The PCR was performed according to Sambrook et al. (Molecular cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press) with an annealing temperature of 55 ° C. The resulting fragment was ligated with the vector pK19mobsac previously cut EcoRI / SalI and treated with alkaline phosphatase. The resulting inactivation vector pK19mobsacBΔpanBC was introduced by transformation into the Escherichia coli strain S 17-1 ( Hanahan (1983) J. Mol. Biol. 166: 557-580 ) and conjugated to Corynebacterium glutamicum ATCC13032 ( Schäfer et al. (1990) J. Bacteriol. 172: 1663-1666 ). Kanamycin-resistant clones of Corynebacterium glutamicum were obtained in which the inactivation vector was integrated in the genome. To select for excision of the vector, kanamycin-resistant clones were seeded on sucrose-containing LB medium (Sambrook et al., Molecular cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press) with 15 g / l agar, 2 % Glucose / 10% sucrose and obtained colonies which have lost the vector by a second recombination event ( Jäger et al. 1992, Journal of Bacteriology 174: 5462-5465 ). By inoculating on minimal medium plates (medium CGXII with 15 g / l agar ( Keilhauer et al., Journal of Bacteriology 175 (1993) 5595-5603 ) with and without 2 mM L-isoleucine or with and without 50 μg / ml kanamycin, 36 clones were isolated which were auxotrophic due to excision of the vector kanamycin sensitive and isoleucine and which now contain the sequence of incomplete pan genes (ΔpanBC alleles ) was present in the genome. One strain was designated ATCC13032ΔpanBC. In the same manner as detailed in this example, the panBC deletion was also introduced into ATCC13032ΔilvA to obtain strain ATCC13032ΔilvAΔpanBC.

Example 5: Expression of the genes ilvD, ilvBN, and ilvC in Corynebacterium glutamicum

The genes of acetohydroxy acid synthase (ilvBN) and isomeroreductase (ilvC) ( Cordes et al. 1992, Gene 112: 113-116 and Keilhauer et al. 1993, Journal of Bacteriology 175: 5595-5603 ) and dihydroxy acid dehydratase (ilvD) (Example 1) were cloned into the vector pECM3 for expression. The vector pECM3 is a derivative of pECM2 ( Jäger et al. 1992, Journal of Bacteriology 174: 5462-5465 ) resulting from deletion of the approximately 1 kbp BamHI / BglII DNA fragment carrying the kanamycin resistance gene.

In the vector pKK5 ( Cordes et al. 1992, Gene 112: 113-116 ) genes ilvBNC were already in the vector pJC1 ( Cremer et al. 1990, Molecular and General Genetics 220: 478-480 ) cloned before. From this, a 5.7 kb XbaIilvBNC fragment was isolated and introduced together with a, containing the ilvD gene, 3.1 kb XbaI fragment of the vector pRV in the XbaI linearized vector pECM3. The ligation mixture was transformed into the E. coli strain DH5αmcr. From one clone, the plasmid pECM3ilvBNCD was obtained.

By electroporation ( Haynes 1989, FEMS Microbiology Letters 61: 329-334 ) and selection for chloramphenicol resistance (3 μg / ml), the plasmid pECM3ilvBNCD was introduced into the strain ATCC13032ΔilvA and the strain ATCC13032ΔilvA / pECM3ilvBNCD was obtained.

Example 6: Production of L-valine with various strains of Corynebacterium glutamicum

To study their valinogenesis, the strains indicated in Table 4 were pre-cultured in 60 ml of Brain Heart infusion medium (Difco Laboratories, Detroit, USA) for 14 h at 30 ° C. Subsequently, the cells were washed once with 0.9% NaCl solution (w / v) and 60 ml of CgXII medium were inoculated with this suspension so that the OD600 was 0.5. The medium was identical to that at Keilhauer et al., (Journal of Bacteriology (1993) 175: 5595-5603 ) described medium. For the cultivation of the ΔilvA strains, however, the medium additionally contained 250 mg / l L-isoleucine. It is shown in Table 3. Table 3 Composition of the medium CGXII component concentration (NH ₄ ) ₂ SO ₄ 20 g / L urea 5 g / L KH ₂ PO ₄ 1 g / L K ₂ HPO ₄ 1 g / L Mg ₂ O ₄ * 7H ₂ O 0.25 g / L 3-morpholino propane 42 g / L CaCl ₂ 10 mg / L FeSO ₄ * 7H ₂ O 10 mg / L MnSO _{4 *} H ₂ O 10 mg / L ZnSO ₄ .7H ₂ O 1 mg / L CuSO ₄ 0.2 mg / L NiCl ₂ .6H ₂ O 0.02 mg / L Biotin (pH7) 0.2 mg / L glucose 40 g / L protocatechuic 0.03 mg / L

After 48 hours of culture, samples were taken, the cells centrifuged off and the supernatant sterile filtered. The L-valine concentration of the supernatant was determined by high pressure liquid chromatography with integrated pre-column derivatization of the amino acid with o-phthdialdehyde as in Jones and Gilligan (J. Chromatogr. 266 (1983) 471-482 ) certainly. The results are shown in Table 4. Table 4 L-valine production with various Corynebacterium glutamicum strains C. glutamicum L-valine (mM) ATCC 13032 0.5 ATCC 13032 pJClilvD 2.2 ATCC 13032 pJC1ilvBNC 20.0 ATCC 13032 pJClilvBNCD 26.2 ATCC 13032 ΔilvA 2.7 ATCC 13032 ΔilvA pJClilvD 7.0 ATCC 13032 ΔilvA pJC1ilvBNCD 28.5 ATCC 13032 ΔpanBC 8.2 ATCC 13032 ΔilvΔΔpanBC 31.1 ATCC 13032 ΔilvAΔpanBC pJC1ilvBNCD 72.7

SEQUENCE LISTING

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Claims (15)

1. Process for the microbial production of L-valine, in which the dihydroxy acid dehydratase (ilvD) activity and/or the ilvD gene expression in a microorganism is intensified, characterised in that the activity of one or more enzymes specifically involved in the synthesis of D-pantothenate is attenuated or turned off.
2. Process according to claim 1, wherein in addition the acetohydroxy acid synthase (ilvBN) activity and isomeroreductase (ilvC) activity and/or ilvBNC gene expression in the microorganism is intensified.
3. Process according to claim 1 or 2, characterised in that the endogenous ilvD or ilvBNCD activity in the microorganism is increased.
4. Process according to claim 3, characterised in that by mutation of the endogenous ilvD gene or ilvBNCD genes corresponding enzymes with higher activity are created.
5. Process according to one or more of the foregoing claims, characterised in that the ilvD or ilvBNCD gene expression is intensified by increasing the gene copy number.
6. Process according to claim 5, characterised in that, to increase the gene copy number, the ilvD gene or the ilvBNCD genes is/are incorporated into a gene construct.
7. Process according to claim 6, characterised in that a microorganism is transformed with the gene construct containing the ilvD gene or ilvBNCD genes.
8. Process according to claim 7, characterised in that Corynebacterium glutamicum is used as the microorganism.
9. Process according to one or more of the foregoing claims, characterised in that a microorganism is used in which the activity of at least one enzyme which is involved in a metabolic path which decreases L-valine formation is attenuated or turned off.
10. Process according to claim 9, characterised in that the activity of the enzyme threonine dehydratase (ilvA) involved in the synthesis of L-isoleucine is attenuated or turned off.
11. Process according to one of claims 1 - 10, characterised in that the activity of the enzyme ketopantoate hydroxymethyl transferase (pan B) and/or the enzyme pantothenate ligase (pan C) is attenuated or turned off.
12. Microorganism transformed with a gene construct containing the ilvD gene or the ilvBNCD genes, wherein the activity of one or more enzymes specifically involved in the synthesis of D-pantothenate is attenuated or turned off.
13. Transformed microorganism according to claim 12, wherein the activity of the enzyme ketopantoate hydroxymethyl transferase (pan B) and/or the enzyme pantothenate ligase (pan C) is attenuated or turned off.
14. Transformed microorganism according to claim 12 or 13, wherein the activity of the enzyme threonine dehydratase (ilvA) involved in the synthesis of L-isoleucine is attenuated or turned off.
15. Transformed microorganism according to one or more of claims 12 to 14, characterised by Corynebacterium glutamicum.

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